Application of KDM6B protein in enhancing cartilage differentiation capability of apical papilla stem cells

Abstract

The invention discloses application of KDM6B protein in enhancing cartilage differentiation capacity of apical papilla stem cells. Experiments prove that KDM6B knocks down and inhibits cartilage differentiation of SCAPs, and overexpresses KDM6B to enhance cartilage differentiation of SCAPs; it is further proved that KDM6B can promote SCAPs mediated cartilage repair, and the effectiveness of KDM6B in cartilage tissue and subosseous bone repair and regeneration is proved in vivo. The invention lays an important foundation for research on treatment of cartilage injury by using the odontogenic stem cells in the future, and has an important application prospect.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and specifically relates to the application of KDM6B protein in enhancing the cartilage differentiation ability of apical papilla stem cells. Background Art

[0002] Articular cartilage is a highly specialized connective tissue that covers the ends of bones in synovial joints. Its main function is to facilitate smooth joint movement and effectively distribute mechanical loads. The unique structure of articular cartilage is composed of sparsely distributed chondrocytes and abundant extracellular matrix (ECM). The ECM is composed of collagen fibers, proteoglycans, and glycosaminoglycans, and the structure of the ECM is essential for maintaining the function of cartilage. However, due to the scarcity of chondrocytes and limited blood supply, articular cartilage is susceptible to damage caused by degenerative diseases, trauma, and aging. These injuries often lead to irreversible structural and functional changes in cartilage, which in turn lead to serious joint diseases such as osteoarthritis. Osteoarthritis is characterized by irreversible abnormalities and dysfunctions in articular cartilage and adjacent tissues, ultimately leading to complete destruction of the joint. Traditional treatments, such as arthroscopic debridement, microfracture surgery, and grinding arthroplasty, are usually unable to effectively restore the integrity and function of cartilage. Therefore, there is an urgent need to develop new and effective strategies to repair articular cartilage.

[0003] In recent years, mesenchymal stem cells (MSCs) have become a promising method for repairing cartilage damage due to their chondrogenic differentiation ability. Dental mesenchymal stem cells such as dental pulp stem cells (DPSCs), human exfoliated deciduous tooth stem cells (shed), periodontal ligament stem cells (PDLSCs) and stem cells from apical papilla (SCAPs) show strong self-renewal and differentiation potential. Among them, SCAPs are superior to DPSCs and PDLSCs in proliferation and differentiation ability and are an ideal cell source for cartilage tissue engineering. However, to date, there is a lack of effective inducing enzymes in the differentiation of SCAPs into chondrocytes, and it is still unclear what epigenetic regulatory factors play a key role in the maturation of chondrocytes and how they affect the cartilage regeneration potential of SCAPs. Therefore, how to enhance the regenerative potential of SCAPs to differentiate into cartilage requires further study.

[0004] KDM6B is a histone demethylase that specifically removes the trimethylation mark of H3K27me3, a modification associated with gene repression. Summary of the invention

[0005] The purpose of the present invention is to promote cartilage regeneration and damage repair.

[0006] The present invention firstly protects the use of KDM6B protein, a substance capable of inducing the expression of KDM6B protein and / or a substance for inhibiting the activity and / or expression of HES1 protein in the preparation of a product; the use of the product is at least one of the following C1) to C8): C1) enhancing cartilage differentiation ability; C2) improving the differentiation potential of chondrocytes; C3) promoting cartilage regeneration; C4) promoting cartilage damage repair; C5) enhancing the cartilage differentiation ability of mesenchymal stem cells; C6) improving the chondrocyte differentiation potential of mesenchymal stem cells; C7) promoting cartilage regeneration of mesenchymal stem cells; C8) promoting cartilage damage repair of mesenchymal stem cells.

[0007] In the above application, the substance used to inhibit the activity and / or expression of HES1 protein can be z1) or z2) or z3): z1) oligonucleotide shRNA; the nucleotide sequence of the oligonucleotide shRNA is shown in SEQ ID NO: 3; z2) shRNA synthesized by the shRNA expression system with the oligonucleic acid shRNA as the target; z3) KDM6B protein.

[0008] In the above application, the shRNA expression system may be a lentiviral expression system.

[0009] In the above application, the z2) can specifically be the plasmid HES1sh mentioned in the embodiment, that is, the plasmid HES1sh (i.e., recombinant lentivirus) constructed by Genema Corporation with HES1 shRNA: 5'-GCTAAGGTGTTTGGAGGCTTC-3' (SEQ ID NO: 3) as the target.

[0010] The present invention also protects the use of KDM6B protein and / or HES1 protein as drug targets in drug screening; the uses of the drug are at least one of the following C1) to C8): C1) enhancing cartilage differentiation ability; C2) improving chondrocyte differentiation potential; C3) promoting cartilage regeneration; C4) promoting cartilage damage repair; C5) enhancing the cartilage differentiation ability of mesenchymal stem cells; C6) improving the chondrocyte differentiation potential of mesenchymal stem cells; C7) promoting cartilage regeneration of mesenchymal stem cells; C8) promoting cartilage damage repair of mesenchymal stem cells.

[0011] In the above application, the HES1 protein as a drug target can be the oligonucleic acid shRNA as a drug target.

[0012] The present invention also protects a product, the active ingredients of which may include KDM6B protein, a substance capable of inducing the expression of KDM6B protein and / or a substance for inhibiting the activity and / or expression of HES1 protein; The uses of the product are at least one of the following C1) to C8): C1) enhancing cartilage differentiation ability; C2) improving chondrocyte differentiation potential; C3) promoting cartilage regeneration; C4) promoting cartilage damage repair; C5) enhancing the cartilage differentiation ability of mesenchymal stem cells; C6) improving the chondrocyte differentiation potential of mesenchymal stem cells; C7) promoting cartilage regeneration of mesenchymal stem cells; C8) promoting cartilage damage repair of mesenchymal stem cells.

[0013] In the above product, the drug may further include a pharmaceutically acceptable carrier.

[0014] In any of the above products, the substance used to inhibit the activity and / or expression of HES1 protein may be z1) or z2) or z3): z1) oligonucleotide shRNA; the nucleotide sequence of the oligonucleotide shRNA is shown in SEQ ID NO: 3; z2) shRNA synthesized by the shRNA expression system with the oligonucleic acid shRNA as the target; z3) KDM6B protein.

[0015] In the above product, the shRNA expression system may be a lentiviral expression system.

[0016] In the above products, the z2) can specifically be the plasmid HES1sh mentioned in the example, that is, the plasmid HES1sh (i.e., recombinant lentivirus) constructed by Jima Gene Corporation with HES1 shRNA: 5'-GCTAAGGTGTTTGGAGGCTTC-3' (SEQ ID NO: 3) as the target.

[0017] Any of the above products may be a medicine.

[0018] Any of the above products may specifically consist of KDM6B protein, a substance capable of inducing the expression of KDM6B protein and / or a substance for inhibiting the activity and / or expression of HES1 protein.

[0019] Any of the above-mentioned substances for inhibiting the activity and / or expression of HES1 protein also falls within the protection scope of the present invention.

[0020] Any of the above-mentioned mesenchymal stem cells may be human dental mesenchymal stem cells.

[0021] The human dental mesenchymal stem cells can be stem cells from apicalpapilla (SCAPs), dental pulp stem cells (DPSCs), human exfoliated deciduous tooth stem cells (shed) or periodontal ligament stem cells (PDLSCs).

[0022] The present invention also protects the use of a substance for detecting the activity and / or expression level of HES1 protein in the preparation of a product for selecting a therapeutic drug for patients with cartilage regeneration and / or cartilage damage repair.

[0023] In the above application, the therapeutic drug may be KDM6B protein.

[0024] The present invention also protects the use of a substance for detecting the activity and / or expression amount of KDM6B protein in the preparation of a product for selecting a therapeutic drug for patients with cartilage regeneration and / or cartilage damage repair.

[0025] Mesenchymal stem cell (MSC)-based therapy has become a promising method for treating articular cartilage damage. However, how to enhance the chondrogenic differentiation potential of MSCs remains a major challenge. This application evaluates the role of KDM6B in the chondrogenic differentiation ability of SCAPs through a large number of experiments (such as Alcian blue staining, pellet culture system or cell transplantation into rabbit knee cartilage defect model). The results showed that KDM6B enhanced the expression of Aggrecan, COL2A1, COL5, glycosaminoglycans and collagen fibers in SCAPs; in vivo, SCAPs overexpressing KDM6B significantly promoted the repair and regeneration of knee cartilage and subchondral bone. At the same time, microarray analysis found that KDM6B regulates the expression of HES1, and further knockdown experiments proved that HES1 is a transcription factor that inhibits the expression of chondrogenic differentiation markers in SCAPs. It can be seen that KDM6B promotes the chondrogenic differentiation potential of SCAPs by inhibiting HES1, providing a potential therapeutic target for enhancing the repair of cartilage damage. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 KDM6B knockdown inhibited the chondrogenic differentiation of SCAPs. Among them, a is the knockdown efficiency of KDM6B in SCAPs detected by Western blot, with histone H3 as the internal reference; bc is the significant inhibition of glycosaminoglycan expression in SCAPs by KDM6B knockdown; df is the downregulation of Aggrecan (d), COL2A1 (e) and COL5 (f) in SCAPs by RT-PCR detection by KDM6B knockdown; g is the staining result of in vitro chondrogenic sphere culture. Statistical significance was determined using Student's t test. All error bars represent standard deviation (SD). (n=3). *P<0.05. **P≤0.01.

[0027] Figure 2KDM6B overexpression enhances chondrogenic differentiation of SCAPs. a is the overexpression efficiency of KDM6B in SCAPs detected by Western blot, with β-actin as an internal reference; bc is the significant increase in the expression of glycosaminoglycans in SCAPs by KDM6B overexpression; df is the upregulation of the expression of Aggrecan (d), COL2A1 (e) and COL5 (f) in SCAPs by RT-PCR detection by KDM6B overexpression; g is the staining result of in vitro chondrogenic sphere culture. Statistical significance was determined using Student's t test. All error bars represent standard deviation (SD). (n=3). *P<0.05. **P≤0.01.

[0028] Figure 3 Figure 2 Macroscopic observation, Micro-CT images, and bone quality analysis results. Among them, ab are the overall view (a) and cross-sectional view (b) of the femoral condyle of the rabbit osteochondral defect model 12 weeks after surgery; c is the Micro-CT three-dimensional image and three-dimensional reconstruction image of three planes (coronal, axial, and sagittal); df is the bone quality analysis of the samples obtained by quantitative Micro-CT analysis, including bone mineral density (BMD) (d), bone volume / total volume (BV / TV) (e), and ICRS score (f). Statistical significance was determined using one-way analysis of variance (ANOVA) or Kruskal-Wallis test. All error bars represent standard deviation (SD). (n=5). *P<0.05. **P≤0.01.

[0029] Figure 4 Histological evaluation of repair tissue after SCAP transplantation. Among them, ab are H&E staining (a) and subosteal bone score (b) of cartilage regeneration; cd are Safranin O staining and cartilage score of regenerated cartilage; ef are immunofluorescence staining for observing COL2 expression. Statistical significance was determined using one-way analysis of variance (ANOVA) or Kruskal-Wallis test. All error bars represent standard deviation (SD). (n=5). *P<0.05. **P≤0.01.

[0030] Figure 5HES1 knockdown enhances chondrogenic differentiation of SCAPs. a is the efficiency of HES1 overexpression in SCAPs detected by Western blot, with β-actin as an internal reference; bc is that HES1 knockdown significantly increased the expression of glycosaminoglycans in SCAPs, with LV3 cells on the left and HES1sh cells on the right in b; df is that HES1 knockdown upregulated the expression of Aggrecan (d), COL2A1 (e) and COL5 (f) in SCAPs detected by RT-PCR; g is the staining result of in vitro chondrogenic sphere culture. Statistical significance was determined using Student's t test. All error bars represent standard deviation (SD). (n=3). *P<0.05. **P≤0.01. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] The histone demethylase KDM6B in the following examples is hereinafter referred to as KDM6B, and its Gene ID is 23135.

[0034] Example 1. Materials and Methods 1. Cell culture SCAPs can be extracted from the apical tissue of patients with immature wisdom teeth or purchased from Shanghai Anwei Biotechnology Co., Ltd. (Shanghai, China). The surface markers of mesenchymal stem cells (MSCs) have been detected. The specific detection methods can be found in the following literature: Zhang C, Ye W, Zhao M, Long L, Xia D, Fan Z. MLL1inhibits the neurogenic potential of SCAPs by interacting with WDR5 andrepressing HES1. Int J Oral Sci. 2023, 15 (1), 48. The culture of SCAPs can be found in the following literature: Zhang C, Ye W, Zhao M, Long L, Xia D, Fan Z. KDM6B Negatively Regulates theNeurogenesis Potential of Apical Papilla Stem Cells via HES1. Int J Mol Sci. 2023, 24 (13). Among them, the steps for self-extraction of SCAPs from the apical tissue of patients with immature wisdom teeth can be as follows: (1) After wisdom teeth were extracted from patients with incomplete root development (all patients gave informed consent), the teeth were immediately rinsed with a large amount of PBS solution. The periapical papilla tissue was carefully removed, cut into pieces, and placed in a digestion solution consisting of an equal volume of 3 mg / mL type I collagenase (Invitrogen, Carlsbad, CA, USA) and 4 mg / mL neutral protease (Invitrogen). The digestion was carried out at 37°C for 45 min, and then the cells were sieved and centrifuged to collect the cells. (2) Resuspend the cells in α-MEM medium containing 15% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin and 2 mmol / l glutamine and inoculate them in a culture dish. Culture at 37°C and 5% CO2, and change the medium every 3 days. (3) When the cells grew to 80% confluence under an inverted microscope, they were digested and passaged with 0.25% trypsin at a ratio of 1:3, added to MSCM medium for culture, and SCAPs were identified; SCAPs of generations 2–4 were used in subsequent experiments.

[0035] 2. Obtaining KDM6B knockdown SCAPs (i.e., KDM6Bsh cells), Scramsh cells, KDM6B overexpressing SCAPs (i.e., HA-KDM6B cells), Vector cells, HES1 knockdown SCAPs (i.e., HES1sh cells), and LV3 cells (1) Obtaining KDM6Bsh cells and Scramsh cells ① Using KDM6B shRNA: 5'-AAGTGGAATGTTTAACAGTGC-3' (SEQ ID NO: 1) as the target and pLKO.1 vector (product of addgene) as the vector, plasmid KDM6Bsh was constructed. Plasmid KDM6Bsh was sequenced, and the sequencing results showed that plasmid KDM6Bsh was a recombinant plasmid obtained by replacing the small DNA fragment between the multiple cloning sites of the pLKO.1 vector with the DNA molecule shown in SEQ ID NO: 1. Plasmid KDM6Bsh knocks down the expression of KDM6B.

[0036] According to the method of step ①, KDM6B shRNA was replaced with knockdown control shRNA: 5'-TTCTCCGAACGTGTCACGTTTC-3' (SEQ ID NO: 2), and other steps remained unchanged to obtain a control plasmid. The control plasmid does not knock down the expression of any protein.

[0037] ②The plasmid KDM6Bsh was transfected into SCAPs, and SCAPs with KDM6B knockdown were screened.

[0038] According to the above steps, the plasmid KDM6Bsh was replaced with the control plasmid, and the knockdown control SCAPs were screened and named Scramsh cells.

[0039] (2) Obtaining HA-KDM6B cells and Vector cells ①Replace the small DNA fragment between the multiple cloning sites of the pQCXIN vector (product of Addgene) with a fusion gene (composed of a hemagglutinin (HA) tag and a gene encoding KDM6B protein from 5' to 3') to obtain the recombinant plasmid pQCXIN-HA-KDM6B. The recombinant plasmid pQCXIN-HA-KDM6B expresses KDM6B.

[0040] ②The recombinant plasmid pQCXIN-HA-KDM6B was transfected into SCAPs, and SCAPs overexpressing KDM6B, namely HA-KDM6B cells, were screened.

[0041] The pQCXIN vector was transfected into SCAPs, and cells carrying the empty vector, namely Vector cells, were screened.

[0042] (3) Obtaining SCAPs (i.e., HES1sh cells) and LV3 cells with HES1 knockdown ① Using HES1 shRNA: 5'-GCTAAGGTGTTTGGAGGCTTC-3' (SEQ ID NO: 3) as the target, we commissioned Genema to construct plasmid HES1sh; then Genema transfected SCAPs with plasmid HES1sh to screen for SCAPs with knockdown of HES1. Plasmid HES1sh knocked down the expression of HES1 (Gene ID 3280).

[0043] ② Using knockdown control shRNA: 5'-TTCTCCGAACGTGTCACGTTTC-3' (SEQ ID NO: 2) as the target, we commissioned Genema to construct a control plasmid; Genema then transfected the control plasmid into SCAPs, screened out knockdown control SCAPs, and named them LV3 cells. The control plasmid did not knock down the expression of any protein.

[0044] In this step, the only difference between the control plasmid and the plasmid HES1sh is the inserted shRNA. The former inserts the knockdown control shRNA, while the latter inserts the HES1 shRNA.

[0045] 3. RT-PCR detection of target genes (such as KDM6Bsh cells, Scramsh cells, LV3 cells, HA-KDM6B cells, Vector cells or HES1sh cells) GAPDH Gene, AGGRECAN Gene, COL5 Gene, COL2A1 Gene or HES1 The relative expression level of genes (1) Total RNA of the target cells was extracted using an RNA extraction kit (Vazyme, RC112-01), and then reverse transcribed into cDNA using a reverse transcription kit (Invitrogen).

[0046] (2) Using the cDNA obtained in step 1 as a template, RT-PCR was performed to detect the relative expression level of the target gene (GAPDH gene was used as an internal reference).

[0047] The RT-PCR detection method specifically refers to the following literature: Zhang C, Ye W, Zhao M, Long L, Xia D, Fan Z. KDM6B Negatively Regulates the Neurogenesis Potential of Apical Papilla Stem Cells via HES1. Int J Mol Sci. 2023, 24 (13). The nucleotide sequences of the primers used to detect the target gene are shown in Table 1.

[0048]

[0049] 4. Western Blot Analysis (1) Extract total protein from target cells (KDM6Bsh cells, Scramsh cells, HA-KDM6B cells or Vector cells), and then perform Western blot analysis to detect the relative expression level of KDM6B protein (using histone H3 as a control).

[0050] The primary antibody used to detect KDM6B protein was KDM6B antibody (1:400, catalog number 07-1533, Millipore, Temecula, CA, USA).

[0051] The primary antibody for detecting Histone H3 was histone H3 antibody (1:1000, catalog number SC-56616, Santa Cruz, CA, USA).

[0052] (2) Extract total protein from target cells (HES1sh cells or LV3 cells), and then perform Western blot analysis to detect the relative expression level of HES1 protein (using β-actin as a control).

[0053] The primary antibody used to detect HES1 protein was HES1 antibody (1:1000, catalog number ab108937, Abcam, UK).

[0054] The primary antibody for detecting β-actin was β-actin antibody (1:2000, catalog number ab6276, Abcam, UK).

[0055] 5. Alcian blue staining and quantitative analysis (1) Seed the target cells (KDM6Bsh cells, Scramsh cells, LV3 cells, HA-KDM6B cells, Vector cells or HES1sh cells) in a 6-well plate at a seeding density of 2 × 10 cells per well. 5The cells were then differentiated into chondrogenic tissues using the StemPro Chondrogenic Differentiation Kit (Invitrogen) for three weeks, during which the culture medium was changed every 3 days; finally, Alcian blue staining was performed.

[0056] The steps of Alcian blue staining are as follows: first, fix the cells with 4% formaldehyde solution at room temperature for 60 minutes, rinse thoroughly with PBS, and stain with 1% Alcian blue solution for 30 minutes; then wash the cells three times with 0.1N HCl to remove unstained areas, and neutralize the pH value with distilled water; then observe the stained cells under an inverted biological microscope, and take images for analysis.

[0057] (2) After completing step (1), quantitative analysis is performed. Specifically, the glycosaminoglycans in the cells are first extracted to obtain extracts; then, the extracts are dissolved in 300 μL 6 M guanidine hydrochloride for 12 hours, and the absorbance at 620 nm is detected using a spectrophotometer.

[0058] 6. Pellet culture system and histological examination (1) Place the target cells (KDM6Bsh cells, Scramsh cells, LV3 cells, HA-KDM6B cells, Vector cells or HES1sh cells) in a centrifuge tube containing culture medium, centrifuge at 1100 rpm for 6 minutes, and loosen the lid; then incubate at 37°C, 5% CO2 for 3 weeks, during which time the culture medium is replaced every 3 days, 0.5 mL each time; obtain primary cell pellets.

[0059] (2) After completing step (1), the primary cell pellet was fixed with 4% paraformaldehyde (PFA) for 24 hours and then embedded in paraffin. The cell pellet was then sectioned (5 µm thick) and subjected to hematoxylin-eosin (H&E) staining, Alcian blue staining (using the Alcian blue staining kit (Cat. No. G2541, Solarbio, Beijing, China)), and Sirius red staining (using the Sirius red staining kit (Cat. No. G1470, Solarbio, Beijing, China)).

[0060] For Alcian blue staining, sections of cell pellets were treated with 3% acetic acid (pH 2.5) for 5 min, followed by staining with 1% Alcian blue for 1 h and counterstained with Fast Nuclear Red for 5 min.

[0061] For Sirius red staining, sections of cell pellets were incubated in 0.1% Sirius red in saturated aqueous picric acid for 1 h and then rinsed in 0.01 M hydrochloric acid for 2 min.

[0062] 7. Rabbit knee cartilage defect creation and transplantation experiment All animal treatments followed the animal care guidelines and regulations of the Animal Experiment Committee of Beijing Stomatological Hospital, Capital Medical University, Beijing, and the animal experiments were approved by the Ethics Committee of Beijing Stomatological Hospital, Capital Medical University, Beijing, with ethics approval number KQYY-202403-001.

[0063] (1) Four transplantation groups were designed based on the factors affecting the transplantation of a mixture of HA-KDM6B cells and hydrogel into the femoral condyle of the rabbit knee joint (see Table 2), namely, sham operation group (Sham), hydrogel group (Matrigel), overexpression control group (Vector) and overexpression experimental group (HA-KDM6B). Five New Zealand rabbits (6 months old, weighing 3 - 3.5 kg) were randomly used in each group. The hydrogel was Matrigel® matrix (Cat. No. 356234, Corning, USA).

[0064]

[0065] (2) After completing step (1), the rabbits were anesthetized by intravenous injection of 3% sodium pentobarbital (35 mg / kg). Under general anesthesia, a standard 5 mm hollow drill was used to create a cylindrical full-thickness osteochondral defect with a diameter of 5 mm and a depth of 4 mm on the medial femoral condyle of rabbits in the hydrogel group, overexpression control group, and overexpression experimental group (refer to the following references for details: Cao Z, Wang H, Chen J, Zhang Y, Mo Q, Zhang P, Wang M, Liu H, Bao X, Sun Y, Zhang W, Yao Q. Silk-based hydrogel incorporated with metal-organicframework nanozymes for enhanced osteochondral regeneration. Bioact Mater. 2023, 20221-242.). Under general anesthesia, the knee joints of rabbits in the sham operation group were opened, and then the knee cartilage was sutured, and no substances, including HA-KDM6B cells, Vector cells, and / or hydrogels, were injected into the knee cartilage.

[0066] (3) After completing step (2), different substances were injected into the defect sites of rabbits in the hydrogel group, overexpression control group, and overexpression experimental group, as shown in Table 1.

[0067] (4) After completing step (3), all rabbits received intramuscular antibiotic injections for 3 days after surgery. Twelve weeks after surgery, all rabbits were euthanized and the femoral condyles were collected for overall observation and further analysis.

[0068] 8. Micro-computed tomography (micro-CT) analysis The femoral condyles of the knee joints of rabbits in the sham, Matrigel, Vector, and HA-KDM6B transplantation groups were scanned using a micro-CT device (Skyscan 1276, Bruker, Germany). After scanning, 3D reconstruction was performed on each knee joint sample. Based on the initial images of the CT data of the four transplantation experimental groups, the coronal, axial, and sagittal reconstruction images of the scanned samples were compared and analyzed. A cylindrical region of interest (ROI) with a diameter of 5 mm and a height of 1.3 mm was selected within the defect regeneration area of ​​each knee joint sample, and the subchondral bone regeneration of each knee joint sample was evaluated by measuring the bone mineral density (BMD) and bone volume to tissue volume ratio (BV / TV) within the ROI (Li P, Fu L, Liao Z, Peng Y, Ning C, Gao C, Zhang D, Sui X, Lin Y, Liu S, Hao C, Guo Q. Chitosan hydrogel / 3D-printed poly(epsilon-caprolactone) hybrid scaffold containing synovialmesenchymal stem cells for cartilage regeneration based on tetrahedralframework nucleic acid recruitment. Biomaterials. 2021, 278 121131.).

[0069] 9. Histological Analysis of Regenerated Cartilage (1) All knee joint specimens were collected and fixed in 10% formalin. After fixation, they were decalcified in 10% EDTA for 2 months. The tissues were then sagittally sectioned, dehydrated, embedded, and cut into 5 µm thick sections.

[0070] (2) After completing step (1), the sections were stained with H&E and Safranin O. The kit used for Safranin O staining was Safranin O staining kit (Cat. No. G1470, Solarbio).

[0071] (3) After completing step (2), immunofluorescence staining was used to detect COL-2 expression in the sections. The antibody used was COL-2 antibody (Cat. No. b-10589R, Bioss, Beijing, China).

[0072] The degree of tissue regeneration was blindly evaluated using the International Cartilage Repair Society (ICRS) scoring system, and the samples were divided into five levels. The specific reference is the following literature: Hoemann C, Kandel R, Roberts S, Saris DB, CreemersL, Mainil-Varlet P, Methot S, Hollander AP, Buschmann MD. International Cartilage Repair Society (ICRS) Recommended Guidelines for Histological Endpoints for Cartilage Repair Studies in Animal Models and Clinical Trials. Cartilage. 2011, 2 (2), 153-172. 10. Statistical analysis To ensure the reliability of the results, each experiment was repeated at least three times. Data analysis was performed using GraphPad5 (GraphPad Software, La Jolla, CA, USA) or SPSS 17 (IBM Corporation, New York, NY, USA). The choice of statistical test (Student's t test, one-way analysis of variance, or Kruskal-Wallis test) depended on the nature and distribution of the data set. In all analyses, a p value ≤ 0.05 was considered statistically significant.

[0073] 2. Experimental Results 1. KDM6B enhances the chondrogenic differentiation potential of SCAPs To knock down KDM6B in SCAPs, we performed lentiviral transfection. After three days of 2µg / mL puromycin treatment, we evaluated the knockdown efficiency of KDM6B by Western blotting. The results showed that the expression of KDM6B protein in KDM6Bsh cells was significantly reduced compared with Scramsh cells ( Figure 1 (a); SCAPs were induced using chondrogenic differentiation medium. Three weeks after induction, Alcian Blue staining and quantitative analysis showed that KDM6Bsh cells formed significantly less glycosaminoglycans than Scramsh cells (see Figure 1b and c); RT-PCR was used to detect the expression of chondrogenic differentiation markers including Aggrecan, COL2A1 and COL5. The results showed that in KDM6Bsh cells, the expression of Aggrecan decreased significantly at two weeks, while the expression of COL2A1 and COL5 decreased significantly at one and two weeks (see Figure 1 To further investigate the effect of KDM6B on the chondrogenic differentiation potential of SCAPs, in vitro chondrogenic sphere culture was performed, and staining results showed that spheres formed by KDM6Bsh cells showed lower levels of glycosaminoglycans and collagen fibers compared with Scramsh cells at three weeks after induction (see Figure 1 Middle g).

[0074] To investigate the effect of KDM6B on the expression of chondrogenic markers in SCAPs, KDM6B was overexpressed in SCAPs, and the efficiency of KDM6B overexpression was analyzed by Western blot. The results showed that the expression of KDM6B protein in HA-KDM6B cells was significantly upregulated compared with that in Vector cells (see Figure 2 (a); SCAPs were induced using chondrogenic differentiation medium. After three weeks of induction, Alcian Blue staining and quantitative analysis showed that HA-KDM6B cells formed significantly more glycosaminoglycans than Vector cells (see Figure 2 b and c); RT-PCR was used to detect the expression of chondrogenic differentiation markers including Aggrecan, COL2A1 and COL5. The results showed that the expression of Aggrecan, COL2A1 and COL5 in HA-KDM6B cells was significantly upregulated at one and two weeks (see Figure 2 In vitro chondrogenic sphere culture results showed that spheres formed by HA-KDM6B cells showed higher levels of glycosaminoglycans and collagen fibers at three weeks after induction compared with Vector cells (see Figure 2 Middle g).

[0075] These results suggest that KDM6B knockdown inhibits the chondrogenic differentiation of SCAPs, while KDM6B enhances the chondrogenic differentiation of SCAPs, that is, KDM6B enhances the chondrogenic differentiation potential of SCAPs.

[0076] 2. KDM6B overexpression promotes SCAP-mediated cartilage regeneration In order to verify the role of KDM6B in cartilage tissue repair and regeneration, this application constructed a knee cartilage model in New Zealand rabbits. At 12 weeks after SCAP transplantation, the in vivo cartilage tissue regeneration was observed. Macroscopic observation showed that the defect areas of the Matrigel group and the Vector group were filled with white new tissue, but the regenerated area was concave. In contrast, the HA-KDM6B group showed excellent cartilage tissue repair, with almost no tissue atrophy in the model area and good integration with the surrounding tissue (see Figure 3 The cross-sectional images showed that a thin layer of translucent tissue appeared in the defect area of ​​the Matrigel group, and the adjacent bone tissue was sparse and porous, resulting in poor integration. In contrast, the defect area of ​​the HA-KDM6B group was filled with transparent repair tissue, which was well integrated with the surrounding bone tissue and almost completely filled the defect area, which performed better than the Matrigel group and the Vector group (see Figure 3 In the analysis of Micro-CT results, significant bone tissue defects were observed in the Matrigel and Vector groups. In sharp contrast, the defect area in the HA-KDM6B group was filled with calcified tissue and well integrated with the surrounding subosteal bone (see Figure 3 c). To further evaluate the subosteal bone regeneration, quantitative analysis was performed, including two-dimensional and three-dimensional reconstruction of rabbit knee joint samples to analyze key indicators such as bone mineral density (BMD) and bone volume to tissue volume ratio (BV / TV). The results showed that 12 weeks after surgery, the bone mineral density of the HA-KDM6B group was significantly higher than that of the Vector group and the Matrigel group, and there was no significant difference compared with the Sham group (see Figure 3 BV / TV evaluation showed that the results of the HA-KDM6B group were significantly improved compared with the other groups at 12 weeks after surgery (see Figure 3 This indicates that the repair of defect tissue in the HA-KDM6B group was enhanced. Under the same conditions, the ICRS score in the HA-KDM6B group was significantly higher than that in the Vector group (see Figure 3 (middle f).

[0077] After SCAP transplantation, the knee joint tissues were further histologically examined. H&E staining results showed that compared with the sham group, a thin cartilage layer was formed in the Matrigel group, and the integration with the subosteal bone and surrounding normal tissue was poor, the cell density was low, and the subosteal bone loss was severe; in the Vector group, the regenerated cartilage tissue had cracks and a chaotic structure; however, in the HA-KDM6B group, a large amount of cartilage tissue was regenerated in the defect area, and its structure was dense and difficult to distinguish from the surrounding healthy tissue in color and texture; in addition, there were a large number of columnar cells in the deep layer of the regenerated tissue, and the cartilage thickness and subosteal bone formation were close to the original tissue (see Figure 4The results of the Modified O'Driscoll scoring system showed that the histological score of the HA-KDM6B group was significantly higher than that of the Vector group and the Matrigel group, and had no significant difference with the Sham group (see Figure 4 b). Safranin O staining results showed that the content of cartilage-like tissue in the Vector group was low and lacked typical chondrocyte morphology; in contrast, the defect area in the HA-KDM6B group was completely filled with cartilage-like tissue and well integrated with the surrounding cartilage; the content of regenerated cartilage matrix was comparable to that in the Sham group (see Figure 4 cd). Immunofluorescence staining showed that the expression of COL2 was weak in the Vector group and Matrigel group; in contrast, the expression of COL2 in the regenerated cartilage tissue of the HA-KDM6B group was strong and well integrated with the surrounding tissue (see Figure 4 In addition, immunofluorescence staining was used to evaluate the changes in COL2 expression. The results showed that the expression of COL2 in the KDM6B group was significantly higher than that in the Matrigel group and the Vector group, and there was no significant difference with the Sham group (see Figure 4 f). It can be seen that overexpression of KDM6B in SCAPs may promote the production of COL2 in the defect site.

[0078] Conclusions: KDM6B promotes SCAP-mediated cartilage regeneration.

[0079] 3. HES1 knockdown enhances chondrogenic differentiation of SCAPs To further clarify the effect of KDM6B on the chondrogenic differentiation of SCAPs, microarray analysis was performed. The results showed that KDM6B effectively inhibited the expression of its downstream target gene HES1. Therefore, the present application also studied the effect of HES1 on the chondrogenic potential of SCAPs.

[0080] To knock down HES1 in SCAPs, we performed lentiviral transfection. After three days of treatment with 2µg / mL puromycin, we evaluated the knockdown efficiency of HES1 by Western blotting. The results showed that the expression of HES1 protein in HES1sh cells was significantly reduced compared with that in LV3 cells ( Figure 5 (a); SCAPs were induced using chondrogenic differentiation medium. Three weeks after induction, Alcian Blue staining and quantitative analysis showed that HES1sh cells formed significantly more glycosaminoglycans than LV3 cells (see Figure 5b and c); RT-PCR was used to detect the expression of chondrogenic differentiation markers including Aggrecan, COL2A1 and COL5. The results showed that in HES1sh cells, the expression of Aggrecan and COL5 was significantly upregulated at two weeks, while the expression of COL2A1 was significantly upregulated at one and two weeks (see Figure 5 In vitro chondrogenic sphere culture results showed that spheres formed by HES1sh cells showed higher levels of glycosaminoglycans and collagen fibers at three weeks after induction compared with LV3 cells (see Figure 5 Middle g).

[0081] Conclusions: HES1 knockdown enhanced the chondrogenic differentiation of SCAPs.

[0082] In summary, KDM6B enhances the differentiation of SCAPs into chondrocytes, and KDM6B can promote SCAPs-mediated cartilage repair, and its effectiveness in the repair and regeneration of cartilage tissue and subosteal bone has been demonstrated in in vivo experiments. Overall, the potential of epigenetic modification in enhancing the ability of SCAPs to promote cartilage repair has been revealed, laying an important foundation for future research on the use of dental stem cells to treat cartilage damage.

[0083] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of KDM6B protein, a substance capable of inducing KDM6B protein expression and / or a substance for inhibiting the activity and / or expression of HES1 protein in the preparation of a product; the use of the product is at least one of the following C1) to C8): C1) enhancing cartilage differentiation ability; C2) improving chondrocyte differentiation potential; C3) promoting cartilage regeneration; C4) promoting cartilage damage repair; C5) enhancing the cartilage differentiation ability of mesenchymal stem cells; C6) improving the chondrocyte differentiation potential of mesenchymal stem cells; C7) promoting cartilage regeneration of mesenchymal stem cells; C8) promoting cartilage damage repair of mesenchymal stem cells.

2. The use according to claim 1, characterized in that: The substance used to inhibit the activity and / or expression of HES1 protein is z1) or z2) or z3): z1) oligonucleotide shRNA; the nucleotide sequence of the oligonucleotide shRNA is shown in SEQ ID NO: 3; z2) shRNA synthesized by the shRNA expression system with the oligonucleic acid shRNA as the target; z3) KDM6B protein.

3. Application of KDM6B protein and / or HES1 protein as drug targets in drug screening; the uses of the drug are at least one of the following C1) to C8): C1) enhancing cartilage differentiation ability; C2) improving chondrocyte differentiation potential; C3) promoting cartilage regeneration; C4) promoting cartilage damage repair; C5) enhancing the cartilage differentiation ability of mesenchymal stem cells; C6) improving the chondrocyte differentiation potential of mesenchymal stem cells; C7) promoting cartilage regeneration of mesenchymal stem cells; C8) promoting cartilage damage repair of mesenchymal stem cells.

4. A product, the active ingredients of which include KDM6B protein, a substance capable of inducing the expression of KDM6B protein and / or a substance for inhibiting the activity and / or expression of HES1 protein; The uses of the product are at least one of the following C1) to C8): C1) enhancing cartilage differentiation ability; C2) improving chondrocyte differentiation potential; C3) promoting cartilage regeneration; C4) promoting cartilage damage repair; C5) enhancing the cartilage differentiation ability of mesenchymal stem cells; C6) improving the chondrocyte differentiation potential of mesenchymal stem cells; C7) promoting cartilage regeneration of mesenchymal stem cells; C8) promoting cartilage damage repair of mesenchymal stem cells.

5. The product according to claim 4, characterized in that: The drug also includes a pharmaceutically acceptable carrier.

6. The product according to claim 4, characterized in that: The substance used to inhibit the activity and / or expression of HES1 protein is z1) or z2) or z3): z1) oligonucleotide shRNA; the nucleotide sequence of the oligonucleotide shRNA is shown in SEQ ID NO: 3; z2) shRNA synthesized by the shRNA expression system with the oligonucleic acid shRNA as the target; z3) KDM6B protein.

7. The use according to claim 1 or 3 or the product according to claim 4, characterized in that: The mesenchymal stem cells are human dental mesenchymal stem cells; Preferably, the dental mesenchymal stem cells are apical papilla stem cells.

8. The substance for inhibiting the activity and / or expression of HES1 protein according to claim 1 or 2.

9. Use of a substance for detecting the activity and / or expression level of KDM6B protein in the preparation of a product for selecting therapeutic drugs for patients requiring cartilage regeneration and / or cartilage damage repair.

10. Use of a substance for detecting the activity and / or expression of HES1 protein in the preparation of a product for selecting a therapeutic drug for patients with cartilage regeneration and / or cartilage damage repair; Preferably, the therapeutic drug is KDM6B protein.

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